Isocratic ion-exchange chromatographic assay for the nucleotide gemcitabine triphosphate in human white blood cells

An isocratic bio-analytical assay for the nucleotide gemcitabine triphosphate (2′,2′-difluorodeoxycytidine 5′-triphosphate, dFdCTP) in human white blood cells (leukocytes) has been developed and validated. The method is based on ion-exchange liquid chromatography and ultraviolet detection (275 nm). dFdCTP is isolated from the matrix by extraction with perchloric acid while the sample is chilled on ice. After neutralization with potassium hydroxide and removal of the potassium perchlorate precipitate, with the sample still chilled on ice, the mixture is injected into the chromatograph. The method has been validated in the range 0.4–20 μM, 0.4 μM (∼20 pmol/106 cells) being the lower limit of quantification, using erythrocytes as a substitute for leukocytes. Precisions and accuracies both meet the current requirements for a bioanalytical assay. The stability of dFdCTP in intact mononuclear blood cells on ice is strongly limited (half-life ∼100 min) and after freezing the half-life of the analyte in the cellular lysate is ∼30 min. On the other hand, no degradation was observed for dFdCTP for at least ∼24 h in perchloric acid extracts on ice or in neutralized extracts at ambient temperature. The applicability of the assay was demonstrated in white blood cells of a patient with advanced non-small cell lung cancer receiving i.v. gemcitabine.